CN112394174B - 一种基于电化学分析用单层MXene增强信号检测心肌肌钙蛋白I的微流体纸基传感器 - Google Patents
一种基于电化学分析用单层MXene增强信号检测心肌肌钙蛋白I的微流体纸基传感器 Download PDFInfo
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Abstract
本发明公开了一种基于单层Mxene修饰的纸基电化学分析装置及方法,属于生物纳米技术领域。该传感器包括检测层A、样品加入层B,所述检测层A用于检测心肌肌钙蛋白cTnI,制作方法:首先在whatman纸上喷蜡打印石蜡,烘烤一定时间形成亲水区域与疏水区域;其次在检测层A丝网印刷碳工作电极,样品加入层B丝网印刷碳对电极以及银‑氯化银参比电极;再次将MXene加入到检测层A的工作电极上,之后将cTnI抗体加入到检测层A的工作电极上,将样品加入层B对折。最后在样品加入层B加入检测样品,利用免疫分析测定,最终通过电化学信号定量检测样品中的cTnI。
Description
技术领域
本发明涉及生物材料、纳米材料,属于生物纳米技术领域,具体涉及基于电化学分析用单层MXene增强信号检测心肌肌钙蛋白I的微流体纸基传感器。
背景技术
2007年,哈佛大学Whitesides团队在纸质检测技术上提出了新的创意。他们基于光刻技术,在纸质衬底表面沉淀疏水聚合物,使纸基具备与预设图样相同的疏水/亲水结构。疏水部分作为壁垒,限制试剂和液体毛细流动范围,亲水部分作为试剂反应区或液体流动通道。Whitesides的创意为健康诊断、环境监测或食品安全等领域的应用提供了一种全新的流体处理和流体分析途径。这种途径最吸引人之处在于:纸质造价低;液体在纸质中流动依靠毛细作用力流动,无需外加动力源;纸基多孔薄膜型结构有筛选、分离等功能;纸质很容易被加工;纸质兼容化学、生化、医学等领域的应用。经过数年的快速发展,纸质微流控传感器已经被视为一种低成本、一次性、易使用和测速快的新型技术。2011年,推出了新的2D MXene纳米材料,与石墨烯相比,具有更复杂的(分层)结构,单层的MXene具有许多特定的性质,如好的亲水性、良好的导电性、丰富的表面基团、大的比表面积和良好的生物相容性,这使它成为电化学检测具有潜力的材料。
急性心肌梗塞(AMI)是威胁生命的严重心血管疾病,为了缓解急性心肌梗塞对人类的危害和提高人们的健康水平,需要研制更灵敏的急性心肌梗塞疾病的检测办法。目前,主要使用酶联免疫吸附实验、电化学发光、免疫比浊法和表面等离子体共振监测患者的AMI,然而这些检测方法只能在医院或专业诊断中心进行,需要使用大型仪器和专业人员操作,因此导致许多人不能得到及时诊断和治疗。而且传统检测方法耗时长、价格昂贵、灵敏度低限制了其实际应用,尤其限制了其在医疗资源匮乏地区的使用。纸基分析装置因其便宜、简单、便携、灵敏有望解决以上难题,开发一种简便廉价、灵敏度高、检测速度快的纸基分析检测新技术有助于早期预防急性心肌梗塞疾病的发生。
发明内容
本发明解决的技术问题是:针对现有技术的不足,提出一种基于电化学分析用单层MXene增强信号检测心肌肌钙蛋白I的微流体纸基传感器,解决了以往检测中样本处理复杂、成本高、不易推广等的缺点。本发明提供了一种成本低、操作简单、灵敏度高、检测限低、快速检测cTnI的纸基电化学传感器。
为了解决上述技术问题,本发明采用如下技术方案:基于电化学分析用单层MXene增强信号检测心肌肌钙蛋白I的微流体纸基传感器,包括以下步骤:
(1)设计纸基模型,在色谱纸上,利用喷蜡打印机打印所需模型,烘烤后,等离子体处理纸;
(2)在步骤(1)检测层A上印刷碳工作电极,在样品加入层B上印刷碳对电极及银-氯化银参比电极;
(3)单层MXene的制备;
(4)单层MXene的硅烷化
(5)心肌肌钙蛋白cTnI抗体的活化;
(6)配制不同浓度的心肌肌钙蛋白cTnI;
(7)在步骤(2)所得的色谱纸的检测层A的工作电极上加入步骤(3)制备的MXene,之后负载心肌肌钙蛋白cTnI抗体;
(8)将步骤(5)中心肌肌钙蛋白cTnI抗原加入到检测层A工作电极上,之后将样品加入层B对折盖在检测层A上进行检测;
(9)利用电化学工作站检测装置的电流信号。
优选的,所述步骤(1)的纸是通过喷蜡打印技术,形成亲水与疏水区域,在100℃左右,烘烤5-10min,再用等离子体清洗器处理纸,通过得失电子,使纸上的羟基变为醛基,醛基再与单层MXene上的氨基反应,以此将单层MXene更好地固定在纸上。
优选的,所述步骤(1)的等离子体处理的纸在加样前需要密封,防止氧化。
优选的,所述步骤(2)的工作电极、对电极和参比电极通过丝网印刷所得。
优选的,所述步骤(3)的MXene的制备方法,将氟化锂与盐酸以质量比1:15混合后加入MXene的前驱体(MAX),搅拌反应24h,离心,清洗多洗,使上层清液呈现墨绿色,之后将多层MXene剥离1h,之后以3500rpm离心1h,得到单层MXene;
优选的,所述步骤(4)的单层MXene需要用三甲氧基硅烷GPTMS处理,以获得能与氨基反应的环氧基团。
优选的,所述步骤(5)的心肌肌钙蛋白cTnI抗体需要用(1-(3-二甲氨基丙基)-3-乙基碳二亚胺·盐酸盐EDC·HCl和N-羟基丁二酰亚胺NHS进行活化。
优选的,所述步骤(7)和(8)的加样和对折,是在色谱纸的检测层A工作电极上加入制备的MXene,反应15-30min后,用磷酸缓冲盐溶液PBS洗涤,之后加入心肌肌钙蛋白cTnI抗体,反应15-30min后,用磷酸缓冲盐溶液PBS洗涤,用牛血清白蛋白BSA阻隔后再洗涤,之后加入心肌肌钙蛋白cTnI抗原,反应15-30min后,用磷酸缓冲盐溶液PBS洗涤,之后将样品加入层B对折盖在检测层A上,加入铁氰化钾和氯化钾。
优选的,所述步骤(9)的电化学检测,通过检测三电极的电流强度,实现定量地检测心肌肌钙蛋白cTnI。
有益效果
与现有技术相比,本发明具有以下优点:
1、本发明的检测心肌肌钙蛋白cTnI的电化学纸基传感器制作方法简单快速,适用范围广。
2、成本低,纸基装置能够降解,对环境无污染。
3、通过更简单方法将抗体活化及负载。
4、运用导电性,电化学稳定性以及生物相容性更好的单层MXene,提高信号。
5、本发明有效地克服了现有技术中本处理复杂、成本高、不易推广等缺点,本发明不仅具有强的特异性、高的灵敏度,还具有低的检测限。
6、本发明提供了一种成本低、操作简单、灵敏度高、检测限低、快速检测cTnI的纸基电化学传感器。
附图说明
图1是本发明的基于电化学分析用MXene增强信号检测心肌肌钙蛋白I的微流体纸基传感器的检测原理图
图2是MXene的原子力显微镜图(AFM)
图3是对抗原抗体结合到工作电极阻抗谱图表征
图4是不同浓度的cTnI的计时电流谱图
图5是纸基检测装置的特异性检测图
具体实施方式
以下上述仅是本发明的具体实施方式,本领域技术人员可通过该说明书所阐述的说明内容轻易地了解本发明的各项细节与应用,在不脱离本发明原理的前提下,还可以做出一些改善,这些改进也应视为本发明的保护范围。
实施例1
基于电化学分析用MXene增强信号检测心肌肌钙蛋白I的微流体纸基传感器,包括以下步骤:
a.等离子体处理纸的形成
1)设计一定大小的纸基模型,通过喷蜡打印技术,形成亲水区域与疏水区域;
2)在90℃下,烘烤5min;
3)用等离子体清洗器处理纸4min得到所需纸;
4)等离子体处理的纸在加样前需要密封,防止氧化。
b.单层MXene的制备
1)5mL水,15mL盐酸与1g氟化锂搅拌混合均匀;
2)加入1g MXene前驱体(MAX)35℃搅拌反应24h;
3)3500rpm离心5min,倒去上清液,反复8次,待上澄层清液变为墨绿色;
4)超声1h,3500rpm离心1h,取清液为单层MXene.
c.单层MXene的硅烷化
1)100mg MXene加入50mL乙醇;
2)加入1mL三甲氧基硅烷GPTMS恒定搅拌(500rpm)48h;
3)室温下搅拌(500rpm)48h,离心;
4)用水和乙醇分别洗涤三遍;
5)50℃下真空干燥12h,研磨,备用。
d心肌肌钙蛋白cTnI抗体的制备
1)将0.2M的EDC·HCl与0.05M的NHS混合;
2)加入50mg/mL的心肌肌钙蛋白cTnI抗体,4℃下活化2h,备用;
e加样并将纸对折;
1)将制备的MXene加到检测层A的工作电极上,反应30min后;
2)用0.01M,pH=7.4的PBS洗涤3次,将活化的心肌肌钙蛋白cTnI抗体加到检测层A的工作电极上
3)用0.01M,pH=7.4的PBS洗涤3次,加入1%BSA阻隔30min,再用0.01M,pH=7.4的PBS洗涤3次;
4)将不同浓度的心肌肌钙蛋白cTnI抗原加到检测层A的工作电极上,反应30min后,用0.01M,pH=7.4的PBS洗涤3次,将检测层A与样品加入层B对折重叠,从样品加入去加入0.05M铁氰化钾和0.2M氯化钾。
f化学检测
1)用电化学工作站检测三电极体系的电流强度,随着cTnI浓度的升高,工作电极的电阻逐渐升高;三电极体系的电流强度,随着cTnI浓度的升高逐渐降低。通过电流强度与cTnI曲线图,就可以实现定量地检测cTnI。
实施例2
基于电化学分析用MXene增强信号检测心肌肌钙蛋白I的微流体纸基传感器,包括以下步骤:
a.等离子体处理纸的形成
1)设计一定大小的纸基模型,通过喷蜡打印技术,形成亲水区域与疏水区域;
2)在100℃下,烘烤5分钟;
3)用等离子体清洗器处理纸4min得到所需纸;
4)等离子体处理的纸在加样前需要密封,防止氧化。
b.单层MXene的制备
1)5mL水,15mL盐酸与1g氟化锂搅拌混合均匀;
2)加入1g MXene前驱体(MAX)35℃搅拌反应24h;
3)3500rpm离心5min,倒去上清液,反复8次,待上澄层清液
变为墨绿色;
4)震荡1h,3500rpm离心1h,取清液为单层MXene.
c.单层MXene的硅烷化
1)100mg MXene加入50mL乙醇;
2)加入1mL三甲氧基硅烷GPTMS恒定搅拌(500rpm)48h;
3)室温下搅拌(500rpm)48h,离心;
4)用水和乙醇分别洗涤三遍;
5)50℃下真空干燥12h,研磨,备用。
d.心肌肌钙蛋白cTnI抗体的制备
1)将0.2M的EDC·HCl与0.05M的NHS混合;
2)加入50mg/mL的心肌肌钙蛋白cTnI抗体,4℃下活化2h,备用;
e.加样并将纸对折;
1)将制备的MXene加到检测层A的工作电极上,反应30min后;
2)用0.01M,pH=7.4的PBS洗涤3次,将活化的心肌肌钙蛋白cTnI抗体加到检测层A的工作电极上
3)用0.01M,pH=7.4的PBS洗涤3次,加入1%BSA阻隔30min,再用0.01M,pH=7.4的PBS洗涤3次;
4)将不同浓度的心肌肌钙蛋白cTnI抗原加到检测层A的工作电极上,反应30min后,用0.01M,pH=7.4的PBS洗涤3次,将检测层A与样品加入层B对折重叠,从样品加入去加入0.05M铁氰化钾和0.2M氯化钾。
f.电化学检测
1)用电化学工作站检测三电极体系的电流强度,随着cTnI浓度的升高,工作电极的电阻逐渐升高;三电极体系的电流强度,随着cTnI浓度的升高逐渐降低。通过电流强度与cTnI曲线图,就可以实现定量地检测cTnI。
实施例3
下面结合附图对本发明的作进一步说明。
图1是本发明的基于电化学分析用MXene增强信号检测心肌肌钙蛋白I的微流体纸基传感器的检测原理。
图2是MXene的原子力显微镜图(AFM)
图3是对抗原抗体结合到工作电极阻抗谱图表征。
图4是不同浓度的cTnI的计时电流谱图。
图5是纸基检测装置的特异性检测图
基于电化学分析用MXene增强信号检测心肌肌钙蛋白I的微流体纸基传感器,包括以下步骤:
a.等离子体处理纸的形成
1)设计一定大小的纸基模型,通过喷蜡打印技术,形成亲水区域与疏水区域;
2)在100℃下,烘烤5分钟;
3)用等离子体清洗器处理纸4min得到所需纸;
4)等离子体处理的纸在加样前需要密封,防止氧化。
b.单层MXene的制备
1)5mL水,15mL盐酸与1g氟化锂搅拌混合均匀;
2)加入1g MXene前驱体(MAX)35℃搅拌反应24h;
3)3500rpm离心5min,倒去上清液,反复8次,待上澄层清液变为墨绿色;
4)震荡1h,3500rpm离心1h,取清液为单层MXene.
c.单层MXene的硅烷化
1)100mg MXene加入50mL乙醇;
2)加入1mL三甲氧基硅烷GPTMS恒定搅拌(500rpm)48h;
3)室温下搅拌(500rpm)48h,离心;
4)用水和乙醇分别洗涤三遍;
5)50℃下真空干燥12h,研磨,备用。
d.心肌肌钙蛋白cTnI抗体的制备
1)将0.2M的EDC·HCl与0.05M的NHS混合;
2)加入50mg/mL的心肌肌钙蛋白cTnI抗体,4℃下活化2h,备用;
e.加样并将纸对折;
1)将制备的MXene加到检测层A的工作电极上,反应30min后;
2)用0.01M,pH=7.4的PBS洗涤3次,将活化的心肌肌钙蛋白cTnI抗体加到检测层A的工作电极上
3)用0.01M,pH=7.4的PBS洗涤3次,加入1%BSA阻隔30min,再用0.01M,pH=7.4的PBS洗涤3次;
4)将不同浓度的心肌肌钙蛋白cTnI抗原加到检测层A的工作电极上,反应30min后,用0.01M,pH=7.4的PBS洗涤3次,将检测层A与样品加入层B对折重叠,从样品加入去加入0.05M铁氰化钾和0.2M氯化钾。
f.电化学检测
1)用电化学工作站检测三电极体系的电流强度,随着cTnI浓度的升高,工作电极的电阻逐渐升高;三电极体系的电流强度,随着cTnI浓度的升高逐渐降低。通过电流强度与cTnI曲线图,就可以实现定量地检测cTnI。
图1为纸基检测装置以及生物样本负载流程。
从图2可以看出,制备的MXene为单层。
从图3可以看出MXene、抗体和抗原被很好的结合到工作电极上。
分别对实施例1中的纸基电化学检测装置用计时电流法进行表征,如图4所示,随着cTnI浓度的升高,电流强度逐渐下降。
从图3可以看出MXene的加入,增加了活性位点增强了电极的导电性,抗原抗体的加入形成一层较薄的绝缘层,降低了导电性。
对实施例1中的不同浓度的cTnI进行电化学的检测,如图4所示,随着cTnI浓度的升高,电流强度逐渐下降,检测范围为10-7-10-2mg/mL,具有低的检测范围。
从图4中电流的强度就可以定量地分析cTnI,并将此方法应用于真实样本。
从图5可以看出纸基检测装置的抗干扰性以及特异性很好。
Claims (9)
1.一种基于电化学分析用单层MXene增强信号检测心肌肌钙蛋白I的微流体纸基传感器,其特征在于:包括以下步骤:
(1)设计纸基模型,在色谱纸上,利用喷蜡打印机打印封闭亲疏水区域,烘烤后,使用等离子清洗机等离子体处理纸;
(2)在步骤(1)检测层A上印刷碳工作电极,在样品加入层B上印刷碳对电极及银-氯化银参比电极;
(3)单层MXene的制备;
(4)单层MXene的硅烷化;
(5)心肌肌钙蛋白cTnI抗体的活化;
(6)配制不同浓度的心肌肌钙蛋白cTnI;
(7)在步骤(2)所得的色谱纸的检测层A的工作电极上加入步骤(3)制备的MXene,之后负载心肌肌钙蛋白cTnI抗体;
(8)将步骤(5)中心肌肌钙蛋白cTnI抗体加入到检测层A工作电极上,之后将样品加入层B对折盖在检测层A上进行检测;
(9)利用电化学工作站检测装置的电流信号。
2.根据权利要求1所述的基于电化学分析用单层MXene增强信号检测心肌肌钙蛋白I的微流体纸基传感器,其特征在于:所述步骤(1)的纸是通过喷蜡打印技术,形成亲水与疏水区域,在100-120℃,烘烤5-10min,再用等离子体清洗器处理纸,通过得失电子,使纸上的羟基变为醛基,醛基再与单层MXene上的氨基反应,以此将单层MXene更好地固定在纸上。
3.根据权利要求1所述的基于电化学分析用单层MXene增强信号检测心肌肌钙蛋白I的微流体纸基传感器,其特征在于:所述步骤(1)的等离子体处理的纸在加样前需要密封,防止氧化。
4.根据权利要求1所述的基于电化学分析用单层MXene增强信号检测心肌肌钙蛋白I的微流体纸基传感器,其特征在于:所述步骤(2)的工作电极、对电极和参比电极通过丝网印刷所得。
5.根据权利要求1所述的基于电化学分析用单层MXene增强信号检测心肌肌钙蛋白I的微流体纸基传感器,其特征在于:所述步骤(3)MXene的制备方法,将氟化锂与盐酸以质量比1:15混合后加入MXene的前驱体MAX,搅拌反应24h,离心,清洗多洗,使上层清液呈现墨绿色,之后将多层MXene剥离1h,之后以3500rpm离心1h,得到单层MXene。
6.根据权利要求1所述的基于电化学分析用单层MXene增强信号检测心肌肌钙蛋白I的微流体纸基传感器,其特征在于:所述步骤(4)的单层MXene需要用三甲氧基硅烷GPTMS处理,以获得能与氨基反应的环氧基团。
7.根据权利要求1所述的基于电化学分析用单层MXene增强信号检测心肌肌钙蛋白I的微流体纸基传感器,其特征在于:所述步骤(5)的心肌肌钙蛋白cTnI抗体需要用(1-(3-二甲氨基丙基)-3-乙基碳二亚胺·盐酸盐EDC·HCl和N-羟基丁二酰亚胺NHS进行活化。
8.根据权利要求1所述的基于电化学分析用单层MXene增强信号检测心肌肌钙蛋白I的微流体纸基传感器,其特征在于:所述步骤(7)和(8)的加样和对折,是在色谱纸的检测层A工作电极上加入制备的单层MXene,反应15-30min后,用磷酸缓冲盐溶液PBS洗涤,之后加入心肌肌钙蛋白cTnI抗体,反应15-30min后,用磷酸缓冲盐溶液PBS洗涤,用牛血清白蛋白BSA阻隔后再洗涤,之后加入心肌肌钙蛋白cTnI,反应15-30min后,用磷酸缓冲盐溶液PBS洗涤,之后将样品加入层B对折盖在检测层A上,加入铁氰化钾和氯化钾。
9.根据权利要求1所述的基于电化学分析用单层MXene增强信号检测心肌肌钙蛋白I的微流体纸基传感器,其特征在于:所述步骤(9)的电化学检测,通过检测三电极的电流强度,实现定量地检测心肌肌钙蛋白cTnI。
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